Project Summary With the advent of routine DNA sequencing of individual tumor and normal tissue samples, opportunities to utilize these data for therapeutic intervention with personalized medicine are currently possible. Insights gained through an understanding of tumor-specific altered DNA repair and tolerance pathways, cell cycle checkpoint control, and intra- and intercellular signaling provide the foundation from which to design combination treatments that leverage mutations and deficiencies in these pathways to achieve synergistic cytotoxicities in tumor versus normal tissues. However, to translate this potential into a clinical reality, it is essential to design treatments that maximize these differential cytotoxic responses. Herein, we propose new DNA adducts that preferentially kill cells that are deficient in homologous recombination (HR), such as those found in BRCA1- and BRCA2-deficient breast and ovarian cancers. The discovery of DNA adducts that arise from the formation of covalent linkage of anthracycline drugs, such as doxorubicin, to exocyclic amino groups in DNA bases, led to a related discovery of novel DNA adducts composed of doxorubicin covalently linked at abasic sites. Strategies are designed that maximize potential therapeutic efficacy through treatments with DNA alkylating agents that rapidly depurinate, leaving abasic sites readily available for secondary reaction with anthracyclines. These first-in-class damages will be engineered into site-specific DNA oligodeoxynucleotides (Project 1) and characterized for their capacity to block DNA replication and RNA transcription. Mechanisms that moderate cytotoxicity such as DNA repair and translesion DNA synthesis will be investigated. Further, cytotoxicities will be optimized in HR-deficient versus proficient cells through selective choice of alkylating agents and temporal delivery of the anthracyclines. Differential cellular responses relative to HR status will be analyzed by tracking alterations in DNA damage response genes. An additional consequence of treatment of cells with alkylating agents is that a subset of these chemotherapeutic agents form interstrand DNA crosslinks. These crosslinks, such as produced by reaction with cyclophosphamide, are also subject to spontaneous decomposition to imidazole ring-opened Fapy-dG adducts and abasic sites in close proximity on complementary strands. These adducts are anticipated to be highly cytotoxic via processing to double-stranded breaks. Analyses of replication and transcription blockage will be performed in addition to analyses of adduct-induced mutagenesis. This approach for combinatorial drug treatments that function via novel DNA adducts to enhance tumor cell killing while using significantly lower doses of drugs, has potential to decrease overall patient dosing and minimize the adverse side-effects of doxorubicin.